JP4735971B2 - Mannosyl erythritol lipid production method - Google Patents
Mannosyl erythritol lipid production method Download PDFInfo
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- JP4735971B2 JP4735971B2 JP2006006305A JP2006006305A JP4735971B2 JP 4735971 B2 JP4735971 B2 JP 4735971B2 JP 2006006305 A JP2006006305 A JP 2006006305A JP 2006006305 A JP2006006305 A JP 2006006305A JP 4735971 B2 JP4735971 B2 JP 4735971B2
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- 238000004519 manufacturing process Methods 0.000 title claims description 71
- -1 Mannosyl erythritol lipid Chemical class 0.000 title claims description 54
- 241001661347 Moesziomyces rugulosus Species 0.000 claims description 47
- 239000004386 Erythritol Substances 0.000 claims description 39
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 claims description 39
- 235000019414 erythritol Nutrition 0.000 claims description 39
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 claims description 39
- 229940009714 erythritol Drugs 0.000 claims description 39
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims description 38
- 235000015112 vegetable and seed oil Nutrition 0.000 claims description 24
- 239000008158 vegetable oil Substances 0.000 claims description 24
- 239000003925 fat Substances 0.000 claims description 22
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 20
- 244000005700 microbiome Species 0.000 claims description 19
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 16
- 239000003921 oil Substances 0.000 claims description 15
- 235000019198 oils Nutrition 0.000 claims description 15
- 238000012258 culturing Methods 0.000 claims description 12
- UJEADPSEBDCWPS-SGJODSJKSA-N (2R,3R)-1-[(3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]butane-1,2,3,4-tetrol Chemical class C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)C([C@H](O)[C@H](O)CO)O UJEADPSEBDCWPS-SGJODSJKSA-N 0.000 claims description 11
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- 229940041514 candida albicans extract Drugs 0.000 claims description 10
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- 239000004317 sodium nitrate Substances 0.000 claims description 10
- 239000012138 yeast extract Substances 0.000 claims description 10
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 9
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 9
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 8
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 8
- 239000013028 medium composition Substances 0.000 claims description 4
- 229940041290 mannose Drugs 0.000 description 34
- 239000002609 medium Substances 0.000 description 31
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 6
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- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
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- HIWPGCMGAMJNRG-ACCAVRKYSA-N Sophorose Natural products O([C@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O)[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HIWPGCMGAMJNRG-ACCAVRKYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
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- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
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- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
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- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
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- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
本発明は、バイオサーファクタントの一種であるマンノシルエリスリトールリピッドの生産効率（生産量、生産速度、及び収率）を大幅に向上させることができるマンノシルエリスリトールリピッドの生産方法に関する。
The present invention relates to a production method of mannosyl erythritol lipid capable of greatly improving the production efficiency (production amount, production rate, and yield) of mannosyl erythritol lipid which is a kind of biosurfactant.
糖脂質は、脂質に１〜数十個の単糖が結合した物質であり、生体内において細胞間の情報伝達に関与し、神経系及び免疫系の機能維持にも重要な役割を果たしていることなどが明らかにされつつある。また、糖脂質は，糖の性質に由来する親水性と脂質の性質に由来する親油性の二つの性質を合わせ持つ両親媒性物質であり、このような性質を有する両親媒性物質は界面活性物質と呼ばれている。石油化学工業が隆盛となるまでは、レシチン、サポニン等の生体成分由来の界面活性剤（バイオサーファクタント）が利用されていた。近年、石油化学工業の発展により合成界面活性剤が開発され、その生産量が飛躍的に増加し、日常生活には無くてはならない物質となったが、この合成界面活性剤の使用量の拡大に伴って環境汚染が広がり、社会問題が生じている。このため、安全性が高く、環境に対する負荷を低減できる生分解性の高い界面活性物質の開発が望まれている。
従来より、微生物が生産する界面活性物質としては、糖脂質系、アシルペプタイド系、リン脂質系、脂肪酸系及び高分子系の界面活性物質の５つに分類されている。これらの中でも、糖脂質系の界面活性剤が最もよく研究されており、細菌及び酵母による多くの種類の界面活性物質が報告されている。
前記細菌としては、Pseudomonas属によるラムノリピッド（非特許文献１及び２参照）とユスチラジン酸（非特許文献３参照）、Rhodococcus属によるトレハロースリピッド（非特許文献４参照）などが知られている。しかし、いずれも生産量は１５ｇ／Ｌ以下である。
前記酵母としては、Candida属によるソホロースリピッドとマンノシルエリスリトールリピッド（特許文献１参照）などが知られている。
前記ソホロースリピッドについては、Candida bombicolaを用いてグルコースとオレイン酸の流加培養法により２００時間で１８０ｇ／Ｌの効率的なソホロースリピッドの生産が可能であることが報告されている（非特許文献５参照）。
Glycolipids are substances in which 1 to several tens of monosaccharides are bound to lipids, are involved in information transmission between cells in vivo, and play an important role in maintaining the functions of the nervous system and immune system. Etc. are being revealed. In addition, glycolipids are amphiphilic substances that have both hydrophilic properties derived from the properties of sugars and lipophilic properties derived from the properties of lipids. It is called a substance. Until the petrochemical industry prospered, surfactants (biosurfactants) derived from biological components such as lecithin and saponin were used. Synthetic surfactants have been developed in recent years due to the development of the petrochemical industry, and their production has dramatically increased, making it an indispensable substance for daily life. As a result, environmental pollution has spread and social problems have arisen. For this reason, it is desired to develop a highly biodegradable surfactant that is highly safe and can reduce the burden on the environment.
Conventionally, the surface active substances produced by microorganisms are classified into five types: surface active substances of glycolipid type, acyl peptide type, phospholipid type, fatty acid type and polymer type. Among these, glycolipid-based surfactants are the most studied, and many types of surfactants from bacteria and yeast have been reported.
Examples of the bacterium include rhamnolipid (see Non-patent Documents 1 and 2) and ustyrazine acid (see Non-Patent Document 3) by Pseudomonas genus, trehalose lipid (see Non-Patent Document 4) by Rhodococcus genus, and the like. However, in all cases, the production amount is 15 g / L or less.
Known yeasts include sophorose lipids and mannosyl erythritol lipids (see Patent Document 1) from the genus Candida .
As for the sophorose lipid, it has been reported that an efficient sophorose lipid of 180 g / L can be produced in 200 hours by the fed-batch culture method of glucose and oleic acid using Candida bombicola (non-patent document). Reference 5).
前記マンノシルエリスリトールリピッド（ＭＥＬ）については、Candida sp.B-7株を用いて５質量％の大豆油から５日間で３５ｇ／Ｌ（生産速度：０．３ｇ／Ｌ／ｈ、原料収率：７０質量％）のＭＥＬの生産が可能であることが報告されている（非特許文献６及び７参照）。また、Candida antarctica T-34株を用いて８質量％の大豆油から８日間で３８ｇ／Ｌ（生産速度：０．２ｇ／Ｌ／ｈ、原料収率：４８質量％）のＭＥＬの生産が可能であることが報告されている（非特許文献８及び９参照）。同じく、Candida antarctica T-34株を用いて６日間隔で計３回の逐次流加により２４日後に２５質量％のピーナッツ油から１１０ｇ／Ｌ（生産速度：０．２ｇ／Ｌ／ｈ、原料収率：４４質量％）のＭＥＬの生産が可能であることが報告されている（非特許文献１０参照）。
Candida sp. SY-16株を用いて１０質量％の植物油脂から回分培養法により２００時間で５０ｇ／Ｌ（生産速度：０．２５ｇ／Ｌ／ｈ、原料収率：５０質量％）のＭＥＬの生産が可能であると共に、流加培養法により２０質量％の植物油から２００時間で１２０ｇ／Ｌ（生産速度：０．６ｇ／Ｌ／ｈ、原料収率：５０質量％）のＭＥＬの生産が可能であることが報告されている（非特許文献１１参照）。
For the mannosyl erythritol lipid (MEL), 35 g / L (production rate: 0.3 g / L / h, raw material yield: 70) from 5% by weight soybean oil using Candida sp. Mass%) MEL is reported to be possible (see Non-Patent Documents 6 and 7). Also, Candida antarctica T-34 strain can be used to produce 38g / L (production rate: 0.2g / L / h, raw material yield: 48% by mass) of 8% by weight soybean oil in 8 days. (See Non-Patent Documents 8 and 9). Similarly, 110 g / L (production rate: 0.2 g / L / h, raw material yield) from 25% by mass of peanut oil after 24 days by continuous feeding 3 times every 6 days using Candida antarctica T-34 strain It has been reported that production of MEL at a rate of 44 mass% is possible (see Non-Patent Document 10).
Using a Candida sp. SY-16 strain , 50 g / L (production rate: 0.25 g / L / h, raw material yield: 50% by mass) of 10 g% of vegetable oil and fat in 200 hours by batch culture method It is possible to produce MEL of 120 g / L (production rate: 0.6 g / L / h, raw material yield: 50 mass%) from 20 mass% vegetable oil in 200 hours by fed-batch culture. It is reported that it is (refer nonpatent literature 11).
Pseudozyma aphidis株を用いて８０質量％の植物油脂から流加培養法により２４時間で１３．９ｇ／Ｌ
（生産速度：０．５７ｇ／Ｌ／ｈ、原料収率：９２質量％）のＭＥＬの生産が可能であることが報告されている（非特許文献１２参照）。
また、醤油醸造工程において副産物として生産されるしょうゆ油（あぶら）を原料としてCandida antarctica T-34株を用いて７日間で８質量％のしょうゆ油から１７ｇ／Ｌ（生産速度：０．１ｇ／Ｌ／ ｈ、原料収率：２１質量％）のＭＥＬの生産が可能であることが提案されている（特許文献２参照）。
Using Pseudozyma aphidis strain , 13.9 g / L from 80% by weight vegetable oil in 24 hours by fed-batch culture method
It has been reported that the production of MEL (production rate: 0.57 g / L / h, raw material yield: 92% by mass) is possible (see Non-Patent Document 12).
17g / L (production rate: 0.1g / L) from 8% by weight of soy sauce oil in 7 days using soy sauce oil (oil) produced as a by-product in the soy sauce brewing process using Candida antarctica T-34 strain / H, raw material yield: 21% by mass) has been proposed (see Patent Document 2).
一方、生分解性が高く、低毒性で環境に優しく、新規な生理機能を持つといわれるマンノシルエリスリトールリピッドなどのバイオサーファクタントを食品工業、医薬品工業、化学工業などで広く普及させていくためには、マンノシルエリスリトールリピッドの生産効率を高め、生産コストの低減を図ることが必要である。 本発明は、このような要望に応え、従来における前記諸問題を解決し、以下の目的を達成することを課題とする。即ち、本発明は、マンノシルエリスリトールリピッドを生産する能力を有する微生物を用い、その培地組成及び培養条件を最適化することによって、マンノシルエリスリトールリピッド（ＭＥＬ）を効率よく生産することができる方法を提供することを目的とする。
On the other hand, in order to widely disseminate biosurfactants such as mannosyl erythritol lipid, which is said to have high biodegradability, low toxicity, environmental friendliness and novel physiological functions, in the food industry, pharmaceutical industry, chemical industry, etc. It is necessary to increase the production efficiency of mannosyl erythritol lipid and to reduce the production cost. An object of the present invention is to meet such demands, solve the above-described problems, and achieve the following objects. That is, the present invention provides a method capable of efficiently producing mannosyl erythritol lipid (MEL) by using a microorganism having the ability to produce mannosyl erythritol lipid and optimizing the medium composition and culture conditions. For the purpose.
前記課題を解決するため本発明者らが鋭意検討を重ねた結果、植物油脂等の油脂類を主成分とするマンノシルエリスリトールリピッド生産用培地においてマンノシルエリスリトールリピッドを生産する能力を有する微生物を培養するに際し、該培地中に、エリスリトール及び／又はマンノースを添加して培養を行うことにより、マンノシルエリスリトールリピッドの生産効率が大幅に上昇することを見出し、本発明を完成するに至った。
すなわち、本発明は以下のとおりである。
As a result of intensive studies by the present inventors in order to solve the above-mentioned problems, when culturing a microorganism having the ability to produce mannosyl erythritol lipids in a medium for producing mannosyl erythritol lipids mainly composed of oils and fats such as vegetable oils and fats, The inventors have found that the production efficiency of mannosyl erythritol lipid is significantly increased by adding erythritol and / or mannose to the medium, thereby completing the present invention.
That is, the present invention is as follows.
（１） 油脂類含有培地でシュードザイマ・ルギュローサ（Pseudozyma rugulosa）に属するマンノシルエリスリトールリピッドを生産する能力を有する微生物を培養してマンノシルエリスリトールリピッドを製造する方法において、上記培地中の初発マンノース濃度を２０〜６０ｇ／Ｌにして培養を行うことを特徴とする、マンノシルエリスリトールリピッドの生産方法。
（２） 培地の初発油脂類濃度が２０〜３００ｇ／Ｌであることを特徴とする、上記（１）に記載の生産方法。
（３） 初発油脂類濃度が２０〜３００ｇ／Ｌ、初発マンノース濃度が２０〜６０ｇ／Ｌ含有する培地で培養を開始し、該培養開始後５〜７日目より油脂類、エリスリトール及び／又はマンノースを培地中に供給することを特徴とする、上記（１）又は（２）に記載のマンノシルエリスリトールリピッドの生産方法。
（４） 培地組成及び培養条件が、以下に示されるものであることを特徴とする、上記（１）〜（３）のいずれかに記載の生産方法。
酵母エキス：０．１〜２ｇ／Ｌ
硝酸ナトリウム：０．１〜１ｇ／Ｌ
リン酸２水素カリウム：０．１〜２ｇ／Ｌ
硫酸マグネシウム：０．１〜１ｇ／Ｌ
マンノース：２０〜６０ｇ／Ｌ
エリスリトール：６０〜８０ｇ／Ｌ
植物油脂：２０〜３００ｇ／Ｌ
培養温度：２６〜３２℃
(1) In a method for producing a mannosyl erythritol lipid by culturing a microorganism having an ability to produce mannosyl erythritol lipids belonging to Pseudozyma rugulosa in an oil and fat-containing medium, the initial mannose concentration in the medium is 20 to A method for producing mannosyl erythritol lipid, characterized by culturing at 60 g / L.
(2) The production method according to (1) above, wherein the initial fat concentration of the medium is 20 to 300 g / L.
(3) Culturing is started in a medium containing an initial oil and fat concentration of 20 to 300 g / L and an initial mannose concentration of 20 to 60 g / L, and oils and fats, erythritol and / or mannose are started from 5 to 7 days after the start of the culture. The method for producing mannosyl erythritol lipid according to the above (1) or (2), characterized in that is supplied in a medium.
(4) The production method according to any one of (1) to (3) above, wherein the medium composition and culture conditions are as follows.
Yeast extract: 0.1-2 g / L
Sodium nitrate: 0.1-1 g / L
Potassium dihydrogen phosphate: 0.1-2 g / L
Magnesium sulfate: 0.1-1 g / L
Mannose: 20-60 g / L
Erythritol: 60-80 g / L
Vegetable oil and fat: 20-300 g / L
Culture temperature: 26-32 ° C
本発明によれば、マンノシルエリスリトールリピッドを生成する能力を有する微生物を植物油脂等の油脂類含有培地で培養するに際し、培地中にマンノース及び／又はエリスリトールを添加することにより、マンノシルエリスリトールリピッドを極めて効率良く生産できる。特に、シュードザイマ属に属する微生物であるシュードザイマ・ルギュローサ（Pseudozyma rugulosa NBRC 10877）を使用する場合には生産量が約２倍に上昇する。このシュードザイマ・ルギュローサ（Pseudozyma rugulosa NBRC 10877）は、従来、マンノシルエリスリトールリピッドを生成する能力を有する微生物としては、知られていなかったものである。しかも、このシュードザイマ・ルギュローサに属する微生物は、マンノース及び／又はエリスリトール添加による生産性向上効果が特に高く、これらを主原料に対し微量添加するだけで、マンノシルエリスリトールリピッドの生産量は約２倍に達する。
また、本発明においては、植物油等の油脂類の消費速度も向上し、結果としてこれら由来の油分の生成物への混入を低減し、マンノシルエリスリトールリピッドの分離精製においても効果が期待できる。
したがって、本発明は、医薬等種々用途への使用が期待されるバイオサーファクタントの生産技術の発展に大いに貢献するものである。
According to the present invention, when a microorganism having the ability to produce mannosyl erythritol lipid is cultured in an oil-containing medium such as vegetable oil, mannose and / or erythritol is added to the medium, thereby making the mannosyl erythritol lipid extremely efficient. Can be produced well. In particular, the amount of production is increased to about twice in the case of using a microorganism belonging to Pseudozyma genus Pseudozyma Rugyurosa (Pseudozyma rugulosa NBRC 10877). The Pseudozyma Rugyurosa (Pseudozyma rugulosa NBRC 10877) is conventionally as a microorganism having the ability to produce mannosylerythritol lipid, in which was not known. Moreover, the microorganism belonging to Pseudozyma rugulosa has a particularly high productivity improvement effect due to the addition of mannose and / or erythritol, and the production amount of mannosyl erythritol lipid reaches about double by simply adding a small amount of these to the main raw material. .
Moreover, in this invention, the consumption rate of fats and oils, such as a vegetable oil, improves, As a result, mixing into the product of the oil component derived from these is reduced, and an effect can be expected also in the separation and purification of mannosyl erythritol lipid.
Therefore, the present invention greatly contributes to the development of biosurfactant production technology expected to be used for various uses such as medicine.
（目的生産物）
本発明の目的生産物であるマンノシルエリスリトールリピッド（ＭＥＬ）は、下記構造式（１）で表される化合物である。
前記マンノシルエリスリトールリピッド（ＭＥＬ）は、高い界面活性作用を有し、界面活性剤又はファインケミカルの種々の触媒として用いられる。ヒト急性前骨髄性白血病細胞性ＨＬ６０株にマンノシルエリスリトールリピッドを作用させると顆粒系を分化させる白血病細胞細胞分化誘導作用があり、 また、ラット副腎髄質褐色細胞腫由来のＰＣ１２細胞にマンノシルエリスリトールリピッドを作用させると神経突起の伸長が生ずる神経系細胞株分化誘導作用等の生理活性作用を有する。更に、微生物産生の糖脂質として初めて、メラノーマ細胞のアポトーシスを誘導することが可能となり（Ｘ． Ｚｈａｏ ｅｔ． ａｌ．， Ｃａｎｃｅｒ Ｒｅｓｅａｒｃｈ，５９， ４８２−４８６（１９９９））、癌細胞増殖抑制作用がある。これらの生理作用から見て、マンノシルエリスリトールリピッドには抗ガン剤等の医薬としての用途が期待される。また、マンノシルエリスリトールリピッド（ＭＥＬ）には生分解性があり、高い安全性を有すると考えられているものである。
(Target product)
Mannosyl erythritol lipid (MEL), which is a target product of the present invention, is a compound represented by the following structural formula (1).
The mannosyl erythritol lipid (MEL) has a high surface activity and is used as various surfactants or fine chemical catalysts. When mannosyl erythritol lipid is allowed to act on human acute promyelocytic leukemia cell line HL60, it induces leukemia cell differentiation that differentiates the granule system, and mannosyl erythritol lipid acts on PC12 cells derived from rat adrenal medullary pheochromocytoma It has a physiological activity such as a neural cell line differentiation-inducing action that causes neurite outgrowth. Furthermore, for the first time as a glycolipid produced by microorganisms, it becomes possible to induce apoptosis of melanoma cells (X. Zhao et. Al., Cancer Research, 59 , 482-486 (1999)) and has an effect of suppressing cancer cell growth. . In view of these physiological actions, mannosyl erythritol lipid is expected to be used as a medicine such as an anticancer agent. In addition, mannosyl erythritol lipid (MEL) is biodegradable and is considered to have high safety.
（使用微生物）
本発明の使用微生物については、マンノシルエリスリトールリピッドを生産する能力を有するものであれば特に制限はなく、例えばシュードザイマ属に属する微生物が挙げられ、このうち特に好ましい微生物としては、シュードザイマ・ルギュローサに属する微生物が挙げることができる。該微生物はマンノシルエリスリトール生産微生物として知られていなかったものであるが、本発明者等により、シュードザイマ・アンタクチカ等の既知のマンノシルエリスリトールリピッド生産菌と同等の生産性を有することが初めて確認されたものである。しかも、このシュードザイマ・ルギュローサに属する微生物は、マンノース及び／又はエリスリトール添加による生産性向上効果が特に高く、これらを主原料に対し微量添加するだけで、マンノシルエリスリトールリピッドの生産量は約２倍に達する点で特筆すべきものである。
(Used microorganism)
The microorganism used in the present invention is not particularly limited as long as it has the ability to produce mannosyl erythritol lipids, and examples thereof include microorganisms belonging to the genus Pseudozyma. Among these, microorganisms belonging to Pseudozyma rugulosa are particularly preferred. Can be mentioned. The microorganism was not known as a mannosyl erythritol-producing microorganism, but was first confirmed by the present inventors to have productivity equivalent to that of known mannosyl erythritol lipid-producing bacteria such as pseudozyma and antactica. It is. Moreover, the microorganism belonging to Pseudozyma rugulosa has a particularly high productivity improvement effect due to the addition of mannose and / or erythritol, and the production amount of mannosyl erythritol lipid reaches about double by simply adding a small amount of these to the main raw material. It is worth mentioning in terms of points.
（マンノシルエリスリトールリピッドの生産）
本発明における使用微生物の培養においては、培地に、脂肪酸、脂肪酸トリグリセリド等の脂肪酸エステル類、あるいは植物油等の油脂類を含有させ、さらにマンノース、エリスリトールを含有させるが、このほかの条件については、特に制限はなく、適宜選定することができる。例えば、酵母に対して一般に用いられる培地を使用でき、このような培地として、例えば、ＹＰＤ培地（イーストイクストラクト１０ｇ、 ポリペプトン２０ｇ、及びグルコース１００ｇ）を挙げることができる。
ＭＥＬの生産量を増加させるためには培地に添加するマンノース及び／又はエリスリトールの供給量を増加させることが好ましく、本発明者等は、培養開始時のマンノース、エリスリトール濃度（初発マンノース、エリスリトール濃度）を変化させて培養を行った結果、培養液中の初発マンノース、エリスリトー ル濃度が少なくとも１％以上、好ましくは４〜８重量％の濃度の場合に、良好なＭＥＬの生産速度、生産量、及び収率が得られるという知見を得ている。これは、生産物すなわちマンノシルエリスリトールリピッドの前駆体物質である原料であるマンノースとエリスリトールを、酵母が細胞内で新たに合成することなく、直接取り込んで炭素源として利用できるため、生産経路が潤滑に進行するためと考えられる。
(Mannosyl erythritol lipid production)
In the culture of the microorganisms used in the present invention, the medium contains fatty acid esters such as fatty acids and fatty acid triglycerides, or fats and oils such as vegetable oils, and further contains mannose and erythritol. There is no restriction and can be selected as appropriate. For example, a medium generally used for yeast can be used, and examples of such a medium include YPD medium (yeast extract 10 g, polypeptone 20 g, and glucose 100 g).
In order to increase the production amount of MEL, it is preferable to increase the supply amount of mannose and / or erythritol added to the medium, and the present inventors have established mannose and erythritol concentrations (initial mannose and erythritol concentrations) at the start of culture. As a result of culturing under various conditions, when the concentration of the initial mannose and erythritol in the culture solution is at least 1%, preferably 4 to 8% by weight, a good MEL production rate, production amount, and The knowledge that the yield is obtained has been obtained. This is because the mannose and erythritol, which are raw materials that are the precursors of the product, mannosyl erythritol lipid, can be directly taken in and used as a carbon source without newly synthesizing it in the cell, so that the production route is lubricated. It is thought to progress.
本発明の使用微生物、特に前記Pseudozyma rugulosa NBRC 10877株を用いてマンノシルエリスリトールリピッドを生産する場合の好適な培地組成及び培養条件は、以下のとおりである。
酵母エキスは、０．１〜２ｇ／Ｌが好ましく、１ｇ／Ｌが特に好ましい
硝酸ナトリウムは、０．１〜１ｇ／Ｌが好ましく、０．５ｇ／Ｌが特に好ましい。
リン酸２水素カリウムは、０．１〜２ｇ／Ｌが好ましく、０．４ｇ／Ｌが特に好ましい。
硫酸マグネシウムは、０．１〜１ｇ／Ｌが好ましく、０．２ｇ／Ｌが特に好ましい。
マンノースは、１ｇ／Ｌ以上が好ましく、２０〜６０ｇ／Ｌが特に好ましい。
エリスリトールは、１ｇ／Ｌ以上が好ましく、２０〜８０ｇ／Ｌが特に好ましい。
植物性油脂は、４０ｇ／Ｌ以上が好ましく、１８０ｇ／Ｌが特に好ましい。
培養温度は、２６〜３２℃が好ましく、３０℃が特に好ましい
The preferred medium composition and culture conditions for producing mannosylerythritol lipids using the microorganisms of the present invention, particularly the Pseudozyma rugulosa NBRC 10877 strain, are as follows.
The yeast extract is preferably 0.1 to 2 g / L, particularly preferably 1 g / L. Sodium nitrate is preferably 0.1 to 1 g / L, particularly preferably 0.5 g / L.
The potassium dihydrogen phosphate is preferably 0.1 to 2 g / L, particularly preferably 0.4 g / L.
Magnesium sulfate is preferably 0.1 to 1 g / L, particularly preferably 0.2 g / L.
Mannose is preferably 1 g / L or more, particularly preferably 20 to 60 g / L.
Erythritol is preferably 1 g / L or more, particularly preferably 20 to 80 g / L.
The vegetable oil is preferably 40 g / L or more, particularly preferably 180 g / L.
The culture temperature is preferably 26 to 32 ° C, particularly preferably 30 ° C.
本発明のマンノシルエリスリトールリピッドの製造方法は、特に制限はなく、目的に応じて適宜選定することができるが、例えば、種培養、本培養及びマンノシルエリスリトールリピッド生産培養の順にスケールアップしていくことが望ましい。
これらの培養における、培地、培養条件を例示すると以下のとおりである。
ａ）種培養；グルコース２０ｇ／Ｌ、酵母エキス１ｇ／Ｌ、硝酸ナトリウム１ｇ／Ｌ、リン酸２水素カリウム ０．５ｇ／Ｌ、及び硫酸マグネシウム０．５ｇ／Ｌの組成の液体培地４ｍＬが入った試験管に１白金耳接種し、３０℃で１日間振とう培養を行う。
ｂ）本培養；所定量の植物性油脂等の油脂類、マンノース及び／又はエリスリトールと、酵母エキス１ｇ／Ｌ、硝酸ナトリウム１ ｇ／Ｌ、リン酸２水素カリウム０．５ｇ／Ｌ、及び硫酸マグネシウム０．５ｇ／Ｌの組成の液体培地１００ｍＬの入った坂口フラスコに接種して、３０℃で２日間培養を行う。
ｃ） マンノシルエリスリトールリピッド生産培養；所定量の植物性油脂等の油脂類、マンノース及び／又はエリスリトールと酵母エキス１ｇ／Ｌ、硝酸ナトリウム１ｇ／Ｌ、リン酸２水素カリウム０．５ｇ／Ｌ、及び硫酸マグネシウム ０．５ｇ／Ｌの組成の液体培地１．４Ｌが入ったジャーファメンターに接種して、３０℃で８００ｒｐｍの撹拌速度で培養を行う。この培養においては、培養途中から植物性油脂、マンノース及び／又はエリスリトールを培養容器中に流下させて、培地中の油脂類濃度を４０〜２００ｇ／Ｌに保持することが望ましい。
以下に、本発明について実施例によりさらに詳細に説明するが、本発明はこれにより限定されるものではない。
The method for producing the mannosyl erythritol lipid of the present invention is not particularly limited and may be appropriately selected according to the purpose. For example, the seed culture, the main culture, and the mannosyl erythritol lipid production culture may be scaled up in this order. desirable.
Examples of culture media and culture conditions in these cultures are as follows.
a) Seed culture: 20 g / L of glucose, 1 g / L of yeast extract, 1 g / L of sodium nitrate, 0.5 g / L of potassium dihydrogen phosphate, and 4 mL of a liquid medium having a composition of 0.5 g / L of magnesium sulfate A platinum loop is inoculated into a test tube and cultured with shaking at 30 ° C. for 1 day.
b) Main culture; predetermined amount of oils and fats such as vegetable oils, mannose and / or erythritol, yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate A Sakaguchi flask containing 100 mL of a liquid medium having a composition of 0.5 g / L is inoculated and cultured at 30 ° C. for 2 days.
c) Mannosyl erythritol lipid production culture; predetermined amount of fats and oils such as vegetable oil, mannose and / or erythritol and yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and sulfuric acid Inoculate a jar fermenter containing 1.4 L of liquid medium having a composition of magnesium 0.5 g / L, and culture at 30 ° C. with a stirring speed of 800 rpm. In this culture, it is desirable that vegetable oil and fat, mannose and / or erythritol are allowed to flow down into the culture vessel from the middle of the culture to maintain the oil and fat concentration in the medium at 40 to 200 g / L.
Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
（Pseudozyma rugulosa NBRC 10877株の培養）
ａ）保存培地（麦芽エキス３ｇ／Ｌ、酵母エキス３ｇ／Ｌ、ペプトン５ｇ／Ｌグルコース１０ｇ／Ｌ、寒天３０ｇ／Ｌ）に保存しておいたPseudozyma rugulosa NBRC 10877株を、 グルコース２０ｇ／Ｌ、酵母エキス１ｇ／Ｌ、硝酸ナトリウムム１ｇ／Ｌ、リン酸２水素カリウム０．５ｇ／Ｌ、及び硫酸マグネシウム０．５ｇ／Ｌの組成の液 体培地４ｍＬが入った試験管に１白金耳接種し、３ ０℃で振とう培養を行い、次いで、
ｂ）得られた菌体培養液を所定量の植物性油脂、マンノース、エリスリトールと酵母エキス１ｇ／Ｌ、硝酸ナトリウム１ｇ／Ｌ、リン酸２水素カリウム０．５ｇ／Ｌ、及び硫酸マグネシウム０．５ｇ／Ｌの組成の液 体培地２０ｍＬの入った坂口フラスコに接種して、３０℃で振とう培養を行い、さらに
ｃ）これを所定量の植物性油脂、マンノース、エリスリトールと酵母エキス１ｇ／Ｌ、硝酸ナトリウム１ｇ／Ｌ、リン酸２水素カリウム ０．５ｇ／Ｌ、及び硫酸マグネシウム０．５ｇ／Ｌの組成の液体培地１．４Ｌが入ったジャーファメンターに接種して、３０℃で８００ｒｐｍの撹拌速度で培養を行った。
上記ａ）〜ｃ）の各培養により得られた菌体培養液を使用して、以下の（１）〜（５）に示される試験を行った。
( Pseudozyma rugulosa NBRC 10877 culture)
a) Pseudozyma rugulosa NBRC 10877 strain stored in a storage medium (malt extract 3 g / L, yeast extract 3 g / L, peptone 5 g / L glucose 10 g / L, agar 30 g / L), glucose 20 g / L, yeast 1 platinum ear is inoculated into a test tube containing 4 mL of a liquid medium composed of 1 g / L extract, 1 g / L sodium nitrate, 0.5 g / L potassium dihydrogen phosphate, and 0.5 g / L magnesium sulfate. Shake culture at 30 ° C., then
b) A predetermined amount of vegetable oil, mannose, erythritol and yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0.5 g Inoculate into a Sakaguchi flask containing 20 mL of a liquid medium having a composition of / L, and carry out shaking culture at 30 ° C., and c) add a predetermined amount of vegetable oil, mannose, erythritol and yeast extract 1 g / L, Inoculate a jar fermenter containing 1.4 L of liquid medium composed of 1 g / L sodium nitrate, 0.5 g / L potassium dihydrogen phosphate, and 0.5 g / L magnesium sulfate, and stir at 30 ° C. at 800 rpm The culture was performed at a rate.
The tests shown in the following (1) to (5) were performed using the bacterial cell culture solutions obtained by the above cultures a) to c).
（試験手法、結果）
（１）バイオサーファクタントの生産の確認
上記ａ）の培養を１日間行った後、ｂ）の培養を、マンノース、エリスルトール無添加で１０日間行い、得られたPseudozyma rugulosa NBRC 10877株の菌体培養液を疎水性のフィルム上にスポットしてその表面張力の変化を観察した。また、コントロールとして培養前の培養液、及び比較例としてPseudozyma antarctica KM-34 (FERMP-20730) 株とPseudozyma aphidisATCC32657株を同様にして培養して得られた培養液をそれぞれスポットして比較した。これらの結果を図１に示す。
これによれば、Pseudozyma rugulosa NBRC 10877株の菌体培養液のスポットにおいては、培地の表面張力がコントロールと同程度低下しており、このことはPseudozyma rugulosa NBRC 10877株がPseudozyma antarctica KM-34(FERMP-20730)株とPseudozyma aphidis ATCC32657株と同様に、バイオサーファクタントが生産可能であることを示している。
(Test method, results)
(1) Confirmation of production of biosurfactant After culturing in a) above for 1 day, culturing in b) was carried out for 10 days without addition of mannose and erythritol, and the resulting cell culture of Pseudozyma rugulosa NBRC 10877 strain Were spotted on a hydrophobic film and the change in surface tension was observed. Moreover, the culture solution before culture | cultivation as a control and the culture solution obtained by culture | cultivating Pseudozyma antarctica KM-34 (FERMP-20730) stock | strain and Pseudozyma aphidis ATCC32657 strain | stump | stock similarly as a comparative example were respectively spotted and compared. These results are shown in FIG.
According to this, in the bacterial culture spot of Pseudozyma rugulosa NBRC 10877, the surface tension of the medium was reduced to the same extent as the control, indicating that Pseudozyma rugulosa NBRC 10877 was Pseudozyma antarctica KM-34 (FERMP -20730) and Pseudozyma aphidis ATCC32657, indicating that biosurfactants can be produced.
（2）Pseudozyma rugulosa NBRC 10877株のマンノシルエリスリトールリピッド（ＭＥＬ）生産能の確認
a）の培養を１日間行った後、ｂ）の培養をマンノース、エリスルトール無添加で１０日間行った後の培養液を採取し、これを用いてPseudozyma rugulosa NBRC 10877株のバイオサーファクタントの生産性を薄層クロマトグラフィーで確認した。一方、比較例としてPseudozyma antarctica KM-34(FERMP-20730) 株を上記と同じ条件で培養し、同様にして薄層クロマトグラフィーを行った。
結果を図２に示す。なお、図中、左端はマンノシルエリスリトールリピッドの標準であり、ＭＥＬ−Ａ，ＭＥＬ−Ｂ及びＭＥＬ−Ｃはそれぞれ順に一般式中（Ｒ１、２＝炭素原子数１〜１４の脂肪酸残基、Ｒ３、４=アセチル基）、同（Ｒ１、２＝炭素原子数１〜１４の脂肪酸残基、Ｒ３=水素原子、Ｒ４=アセチル基）及び同（Ｒ１、２＝炭素原子数１〜１４の脂肪酸残基、Ｒ３=アセチル基、Ｒ４=水素原子）で表される化合物を示す。
これによれば、両株とも大豆油含有培地でマンノシルエリスリトールリピッド（MEL）を生産している。また、マンノシルエリスリトールリピッドの標準と対比して、Pseudozyma rugulosa NBRC 10877株が生産しているバイオサーファクタントはマンノシルエリスリトールリピッドであることがわかる。
(2) Confirmation of Pseudozyma rugulosa NBRC 10877 production ability of mannosyl erythritol lipid (MEL)
After the culture in a) is performed for 1 day, the culture solution after b) is cultured for 10 days without adding mannose and erythritol is collected and used to increase the productivity of the biosurfactant of Pseudozyma rugulosa NBRC 10877. Confirmed by thin layer chromatography. On the other hand, as a comparative example, Pseudozyma antarctica KM-34 (FERMP-20730) strain was cultured under the same conditions as described above, and thin layer chromatography was performed in the same manner.
The results are shown in FIG. In the figure, the left end is a standard of mannosyl erythritol lipid, and MEL-A, MEL-B and MEL-C are respectively in the general formula (R 1 , 2 = fatty acid residue having 1 to 14 carbon atoms, R 3 , 4 = acetyl group), (R 1 , 2 = fatty acid residue having 1 to 14 carbon atoms, R 3 = hydrogen atom, R 4 = acetyl group) and the same (R 1 , 2 = carbon atom number 1) -14 fatty acid residues, R 3 = acetyl group, R 4 = hydrogen atom).
According to this, both strains produce mannosyl erythritol lipid (MEL) in a soybean oil-containing medium. In addition, it can be seen that the biosurfactant produced by Pseudozyma rugulosa NBRC 10877 is mannosyl erythritol lipid as compared with the standard of mannosyl erythritol lipid.
（３）マンノシルエリスリトールリピッド（ＭＥＬ）生産用培地で同リピッドの生産
Pseudozyma rugulosa NBRC 10877株を用い、ａ）の培養を１日間行った後、ｂ）の培養をマンノース、エリスルトール無添加で１０日間行った。培養液を採取し、そのＭＥＬ生産量を高速液体クロマトグラフィーで検出した。また、比較例としてPseudozyma antarctica KM-34(FERMP-20730)株を上記と同じ条件で培養し、同様にして高速液体クロマトグラフィーで検出した。結果を図３に示す。なお、図３は、培養液中の酢酸エチル可溶分を高速液体クロマトグラフィーで検出した結果であり、既知のマンノシルエリスリトールリピッドのものと一致する。図３によれば、Pseudozyma rugulosa NBRC 10877株はPseudozyma antarcticaKM-34(FERMP-20730)株と同程度のMEL生産能力を有する。
(3) Mannosyl erythritol lipid (MEL) production in the same medium
Using Pseudozyma rugulosa NBRC 10877 strain, a) was cultured for 1 day, and then b) was cultured for 10 days without adding mannose and erythritol. The culture solution was collected, and the production amount of MEL was detected by high performance liquid chromatography. In addition, as a comparative example, Pseudozyma antarctica KM-34 (FERMP-20730) strain was cultured under the same conditions as above and detected by high performance liquid chromatography in the same manner. The results are shown in FIG. FIG. 3 shows the result of detection of ethyl acetate-soluble components in the culture solution by high performance liquid chromatography, which is consistent with that of known mannosyl erythritol lipids. According to FIG. 3, the Pseudozyma rugulosa NBRC 10877 strain has the same MEL production capacity as the Pseudozyma antarctica KM -34 (FERMP-20730) strain.
（４）Pseudozyma rugulosa NBRC 10877株が生産するマンノシルエリスリトールリピッド（ＭＥＬ）の構造解析
Pseudozyma rugulosa NBRC 10877株を用い、ａ）の培養を１日間行った後、ｂ）の培養をマンノース、エリスルトー ル無添加で１０日間行った。培養液を採取し、培養液中のＭＥＬを酢酸エチルで抽出し、１H NMR で生産物の構造解析を行った（上段）。また、典型的なMELの構造解析結果の例としてPseudozyma antarctica KM-34(FERMP-20730)株が生産するMELの分析結果を下段に示す。図４によれば、Pseudozyma rugulosa NBRC 10877株が生産する物質は典型的なMELの構造を有することが明らかである。
(4) Structural analysis of mannosylerythritol lipid (MEL) produced by Pseudozyma rugulosa NBRC 10877
Using Pseudozyma rugulosa NBRC 10877 strain, a) was cultured for 1 day, and then b) was cultured for 10 days without adding mannose and erythritol. The culture solution was collected, MEL in the culture solution was extracted with ethyl acetate, and the structure of the product was analyzed by 1 H NMR (top). In addition, as an example of a typical structural analysis result of MEL, an analysis result of MEL produced by Pseudozyma antarctica KM-34 (FERMP-20730) strain is shown in the lower part. According to FIG. 4, it is clear that the substance produced by Pseudozyma rugulosa NBRC 10877 has a typical MEL structure.
（５）Pseudozyma rugulosa NBRC 10877株のMEL生産に対する窒素源の影響
Pseudozyma rugulosa NBRC 10877株を用い、ａ）の培養を１日間行った後、b）の培養をマンノース、エリスリトール無添加で１０日間行った後、培養液を採取し、そのＭＥＬ生産量を高速液体クロマトグラフィーで検出した。このとき、培養液中の窒素源はそれぞれ図中に記載した組成に調製したものを用いた。結果を図５に示す。この結果によれば、Pseudozyma rugulosa NBRC 10877株によるMEL生産において、最適な窒素源は硝酸ナトリウムである。
(5) Effect of nitrogen source on MEL production of Pseudozyma rugulosa NBRC 10877
Using Pseudozyma rugulosa NBRC 10877, a) was cultured for 1 day, b) was cultured for 10 days with no mannose and erythritol added, and the culture was collected, and the amount of MEL produced was measured by high performance liquid chromatography. Detected by graphy. At this time, the nitrogen sources in the culture solution were each prepared to have the composition described in the figure. The results are shown in FIG. According to this result, sodium nitrate is the most suitable nitrogen source for MEL production by Pseudozyma rugulosa NBRC 10877.
（６）Pseudozyma rugulosa NBRC 10877株のMEL生産に対する植物性油脂の影響
Pseudozyma rugulosa NBRC 10877株を用い、ａ）の培養を１日間行った後、b）の培養をマンノース、エリスリトール無添加で１０日間行った後、培養液を採取し、そのＭＥＬ生産量を高速液体クロマトグラフィーで検出した。このとき、培養液中の植物性油脂はそれぞれ図中に記載した組成に調製したものを用いた。結果を図６に示す。この結果によれば、Pseudozyma rugulosa NBRC 10877株は各種植物性油脂からMELを生産し、大豆油が最も良好な結果を示した。
(6) Effects of vegetable oils and fats on MEL production of Pseudozyma rugulosa NBRC 10877
Using Pseudozyma rugulosa NBRC 10877, a) was cultured for 1 day, b) was cultured for 10 days with no mannose and erythritol added, and the culture was collected, and the amount of MEL produced was measured by high performance liquid chromatography. Detected by graphy. At this time, vegetable oils and fats in the culture solution were prepared to the compositions described in the figure. The results are shown in FIG. According to this result, Pseudozyma rugulosa NBRC 10877 produced MEL from various vegetable oils and fats, and soybean oil showed the best results.
（７）Pseudozyma rugulosa NBRC 10877株のMEL生産に対する水溶性炭素源の影響
Pseudozyma rugulosa NBRC 10877株を用い、ａ）の培養を１日間行った後、b）の培養をマンノース８０ｇ／Ｌ、エリスリトール８０ｇ／Ｌ、あるいはグルコース８０ｇ／Ｌをそれぞれ添加して各々１０日間行った後、培養液を採取し、そのＭＥＬ生産量を高速液体クロマトグラフィーで検出した。このとき、培養液中に植物性油脂として大豆油４０ｇ／Ｌを添加した。結果を図７に示す。この結果によれば、Pseudozyma rugulosa NBRC 10877株によるMELの生産量はエリスリトールを添加したとき約２倍上昇した。
(7) Effect of water-soluble carbon source on MEL production of Pseudozyma rugulosa NBRC 10877
After culturing a) for 1 day using Pseudozyma rugulosa NBRC 10877 strain, after culturing b) for 10 days by adding mannose 80 g / L, erythritol 80 g / L, or glucose 80 g / L, respectively. The culture solution was collected, and the production amount of MEL was detected by high performance liquid chromatography. At this time, 40 g / L of soybean oil was added as a vegetable oil to the culture solution. The results are shown in FIG. According to this result, the production amount of MEL by the Pseudozyma rugulosa NBRC 10877 strain increased about 2-fold when erythritol was added.
（８）Pseudozyma rugulosa NBRC 10877株のMEL生産に対するエリスリトール濃度の影響
Pseudozyma rugulosa NBRC 10877株を用い、ａ）の培養を１日間行った後、b）の培養を図８に記載の各濃度でエリスリトールを添加して１０日間行った後、培養液を採取し、そのＭＥＬ生産量を高速液体クロマトグラフィーで検出した。このとき、培養液中に植物性油脂として大豆油４０ｇ／Ｌを添加した。結果を図８に示す。なお、数値はエリスリトール無添加時の生産量を１００とした時の相対地として示している。この結果によれば、Pseudozyma rugulosa NBRC 10877株によるMELの生産量はエリスリトール２０ｇ／Ｌ以上を添加したとき約２倍上昇した。
(8) Effect of erythritol concentration on MEL production of Pseudozyma rugulosa NBRC 10877
Pseudozyma rugulosa NBRC 10877 strain was used, a) was cultured for 1 day, b) was cultured for 10 days with erythritol added at each concentration shown in FIG. MEL production was detected by high performance liquid chromatography. At this time, 40 g / L of soybean oil was added as a vegetable oil to the culture solution. The results are shown in FIG. In addition, a numerical value is shown as a relative place when the production amount when no erythritol is added is 100. According to this result, the production amount of MEL by Pseudozyma rugulosa NBRC 10877 was increased about twice when erythritol 20 g / L or more was added.
（９）Pseudozyma rugulosa NBRC 10877株のＭＥＬ生産に対するマンノース濃度の影響
Pseudozyma rugulosa NBRC 10877株を用い、ａ）の培養を１日間行った後、ｂ）の培養を図９に記載の濃度でマンノースを添加して１０日間行った後、培養液を採取し、そのＭＥＬ生産量を高速液体クロマトグラフィーで検出した。このとき、培養液中に植物性油脂として大豆油４０ｇ／Ｌを添加した。結果を図９に示す。なお、数値はマンノース無添加時の生産量を１００とした時の相対値として示している。この結果によれば、Pseudozyma rugulosa NBRC 10877株によるMELの生産量はマンノース２０〜６０ｇ／Ｌを添加したとき約１．７倍上昇した。マンノース８０ｇ／Ｌの添加では生産量の上昇効果が低下した。
(9) Effect of mannose concentration on MEL production of Pseudozyma rugulosa NBRC 10877
Using Pseudozyma rugulosa NBRC 10877 strain, after 1 day of culture in a), after 10 days with the addition of mannose at a concentration according to culture b) in FIG. 9, taken culture, the MEL The production amount was detected by high performance liquid chromatography. At this time, 40 g / L of soybean oil was added as a vegetable oil to the culture solution. The results are shown in Figure 9. In addition, a numerical value is shown as a relative value when the production amount when no mannose is added is 100. According to this result, the production amount of MEL by Pseudozyma rugulosa NBRC 10877 increased about 1.7 times when mannose 20-60 g / L was added. Addition of mannose 80 g / L decreased the production increase effect.
（１０）Pseudozyma rugulosa NBRC 10877株のMEL生産培養
上記ａ）の培養を１日間行った後、ｂ）の培養を、マンノース、エリスルトール無添加で１０日間行い、続いて、ｃ）の培養を行った。ｃ）の培養は８０ｇ/Lの植物性油脂と２０ｇ/Lあるいは８０ｇ/Lのエリスリトールを含む培地を用いて行った。培養途中から８０ｇ/Lの植物性油脂と所定量のエリスリトールを１週間毎に培養容器中に流下させた。図１０は、１週間毎に培養液を採取し、ＭＥＬ生産量を高速液体クロマトグラフィーで定量して作製したグラフである。矢印は炭素源を添加した時点を示している。比較の対象として、エリスリトールを含まない培地で生産培養した結果を用いた。図１０によると、２％あるいは８％のエリスリトールを培地中に加えた場合、２８日間で生産されたMELの量は１９０ｇ/L以上に達しており、エリスリトールを加えない条件の約３倍上昇した。このときの生産収率は約７９％であった。
(10) MEL production culture of Pseudozyma rugulosa NBRC 10877 strain After the culture of the above a) was performed for 1 day, the culture of b) was performed for 10 days without addition of mannose and erythritol, and then the culture of c) was performed. . The culture in c) was performed using a medium containing 80 g / L vegetable oil and fat and 20 g / L or 80 g / L erythritol. From the middle of the culture, 80 g / L of vegetable oil and fat and a predetermined amount of erythritol were allowed to flow down into the culture vessel every week. FIG. 10 is a graph prepared by collecting a culture solution every week and quantifying the MEL production amount by high performance liquid chromatography. The arrow indicates the time when the carbon source is added. As a comparison target, the results of production and culture in a medium not containing erythritol were used. According to FIG. 10, when 2% or 8% erythritol was added to the medium, the amount of MEL produced in 28 days reached 190 g / L or more, which was about three times higher than the condition without adding erythritol. . The production yield at this time was about 79%.
Claims (4)
酵母エキス：０．１〜２ｇ／Ｌ
硝酸ナトリウム：０．１〜１ｇ／Ｌ
リン酸２水素カリウム：０．１〜２ｇ／Ｌ
硫酸マグネシウム：０．１〜１ｇ／Ｌ
マンノース：２０〜６０ｇ／Ｌ
エリスリトール：６０〜８０ｇ／Ｌ
植物油脂：２０〜３００ｇ／Ｌ
培養温度：２６〜３２℃
The production method according to any one of claims 1 to 3, wherein the medium composition and the culture conditions are as follows.
Yeast extract: 0.1-2 g / L
Sodium nitrate: 0.1-1 g / L
Potassium dihydrogen phosphate: 0.1-2 g / L
Magnesium sulfate: 0.1-1 g / L
Mannose: 20-60 g / L
Erythritol: 60-80 g / L
Vegetable oil and fat: 20-300 g / L
Culture temperature: 26-32 ° C
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